Abstract:
The aim of the present study was to identify chemical composition and evaluate antimicrobial effectiveness of the essential oil of Cistus ladanifer var. maculatus Dun. The essential oil of C. ladanifer’s leaves was obtained by hydro-distillation and analyzed by gas chromatography coupled with mass spectrometry (GC/MS). 28 compounds were identified, representing 88.67% of the total constituents. The main compound was identified as Verticiol (18.16%), followed by Camphene (17.70%), γ-Gurjunene (7.15%), n-Butylcyclohexane (5.95%) and Bornyl acetate (5.86%). The oil was found to be rich in Monoterpene Hydrocarbons (32.05%), followed by Diterpenic alcohol (18.16%), Oxygenated hydrocarbons (13.27%), Sesquiterpene hydrocarbons (12.40%), Monoterpenic ester (5.86%), Cyclic ether (4.36%) and Oxygenated sesquiterpenes (2.57%).
Antimicrobial activity of the essential oil of C. ladanifer was tested against two Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, and three fungi (Candida albicans, Trichophyton rubrum and Aspergillus niger), by the microdilution method. In fact, strong inhibitory and bactericidal effect has been shown against all tested Gram-positive and Gram-negative bacteria. Indeed, strongest inhibitory and fungicide effect was exerted against the yeasts and molds studied.
It seems following this study that the essential oil of C. Ladanifer has very significant antimicrobial activity, it shows a broad spectrum covering Gram-positive, Gram-negative bacteria, yeasts and fungi.

AUTHORS

ABSTRACT

The aim of the present study was to identify chemical composition and evaluate antimicrobial effectiveness of the essential oil of Cistus ladanifer var. maculatus Dun. The essential oil of C. ladanifer’s leaves was obtained by hydro-distillation and analyzed by gas chromatography coupled with mass spectrometry (GC/MS). 28 compounds were identified, representing 88.67% of the total constituents. The main compound was identified as Verticiol (18.16%), followed by Camphene (17.70%), γ-Gurjunene (7.15%), n-Butylcyclohexane (5.95%) and Bornyl acetate (5.86%). The oil was found to be rich in Monoterpene Hydrocarbons (32.05%), followed by Diterpenic alcohol (18.16%), Oxygenated hydrocarbons (13.27%), Sesquiterpene hydrocarbons (12.40%), Monoterpenic ester (5.86%), Cyclic ether (4.36%) and Oxygenated sesquiterpenes (2.57%).
Antimicrobial activity of the essential oil of C. ladanifer was tested against two Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, and three fungi (Candida albicans, Trichophyton rubrum and Aspergillus niger), by the microdilution method. In fact, strong inhibitory and bactericidal effect has been shown against all tested Gram-positive and Gram-negative bacteria. Indeed, strongest inhibitory and fungicide effect was exerted against the yeasts and molds studied.
It seems following this study that the essential oil of C. Ladanifer has very significant antimicrobial activity, it shows a broad spectrum covering Gram-positive, Gram-negative bacteria, yeasts and fungi.

KEYWORDS

For centuries, medicinal plants have been used as a remedy for various diseases. Plants have the capability to synthesize a wide range of phytochemicals that possess significant biological functions. Many of these compounds have beneficial effects on human health and can be used to treat diseases (Mahesh and Satish, 2008; Prasannabalaji et al., 2012).

Moreover, plants have the ability to synthesize aromatic substances, many of which are bioactive phytochemicals (Cowan, 1999). Over the past few years, the use of plant based natural antimicrobials in the treatment of bacterial infections has gained much recognition (Kasrati et al., 2014).

Furthermore, antimicrobial resistance is now a global concern, which reached a crisis point in many hospitals worldwide. Indeed, there is an urgent need to replace our arsenal of anti-infective agents (Cushnie, 2011), by developing new antimicrobial agents. In this context, a great interest has been focused on the natural products, in particular essential oils (Bakkali et al., 2008). One of their apparent properties is their antiseptic power, linked to their activity against pathogenic bacteria, including some antibiotic resistant strains. Several previous data already described many recipes of herbal and aromatic oils that priests and doctors employed. Currently, the use of essential oils is carried out on scientific and rational basis, in order to develop new products for various fields: food, medical, veterinary and cosmetic.

Hence, several research studies have proved the antimicrobial, antiviral and insecticide properties of terpenes (Salah-Fatnassi et al., 2010; Nsambu et al., 2014). In addition, chemical complexity of the essential oils prevents the decoding from pathogens and thus reduces the risk of developing microbial resistance.

Sixteen species of the genus Cistus (Cistaceae) are known to be native in the Mediterranean area’s flora (Gültz et al., 1996). Leaves of all Cistus taxa are covered with glands secreting resin and essential oil. Plants essential oil and resin consist mainly of terpenoids (Gültz et al., 1984; Demetzos et al., 1994c), flavonoid aglycons (Vogt et al., 1987b; Demetzos et al., 1990), and glycosides (Vogt et al., 1987a). Labdane diterpenes, which appears to be the predominant compounds of the essential oils have been studied for their antimicrobial and cytotoxic activity (Chinou et al., 1994; Demetzos et al., 1994a; Demetzos et al., 1994b; Dimas et al., 1998; Anastassaki et al., 1999). In the family Cistaceae, Cistus ladanifer is the most important species used in the perfume and cosmetic industries (Mariotti et al., 1994). C. ladanifer is a very poorly exploited species in Morocco for its aromatic virtues; although it is famous on the Mediterranean market (Spain and France). Indeed, Spain is the main producer of the oil which is obtained from the leafy branches, while labdanum extracts are obtained from the gum (Simon-Fuentens et al., 1987). C. ladanifer is represented in Morocco by two varieties which differ primarily by the flowers petal color: C. ladanifer var. albiflorus Dun with completely white petals and C. ladanifer var. maculatus Dun with mottled petals of crimson. This present study was performed on var. maculatus which grows spontaneously in the Moroccan Middle Atlas.

In this study, we examined the chemical composition of volatile components and antimicrobial activity of the leaves oil. The volatile components were identified using GC/MS. The antimicrobial activity was studied by microdilution methods against 7 selected degrading and pathogenic microorganisms.

MATERIAL AND METHODS

Plant material

The aerial part (stems, leaves) of C. ladanifer var. maculatus Dun were collected in August 2013 in the region of Harcha (Oulmes, Morocco). The Harcha forest spreads out on a mountainous area. It is characterized by a subhumid region, surrounded by a semi-arid zone and is located between 600 and 1300 m of altitude (Boudy, 1951). The botanical identification was performed by Dr. Ahmed BOUKIL (Chief Engineer of Water and Forests, UNDP consultant).

Isolation of essential oil

From fresh leaves of C. ladanifer var. maculatus Dun, 200g was hydrodistilled for 5 hours using a Clevenger-type apparatus. The essential oil was collected, dried over anhydrous Na2SO4, and was then kept in dark at 4°C until further use.

Chemical analysis

Analysis of the volatile compounds was performed on Hewlett-Packard gas chromatograph/mass spectra (GC/MS) system. The chromatograph in split ratio (1:30) mode was fitted with a DB-5 fused silica capillary column (30 m x 0.25 mm, film thickness 0.25 mm). The carrier gas was He with a flow rate close to 2 mL/min. The injector and detector temperatures were 250°C and 280°C, respectively. The column temperature was programmed as follows: 50°C (10 min), 50-190°C at (2°C/min) and 190°C (10 min). The mass spectra were recorded on a selective quadrupolar type detector of 5772 class, ionization was obtained by electronic impacts under a potential of 70 eV. The mass range was between m/z 35-425 amu, while scan time was 0.2 scan/s. Compounds were identified by their mass spectra and retention indices. Quantitative analysis was conducted on a HP 5890 gas chromatograph fitted with a flame ionization detector. The column and conditions of use were the same; although N was used as the carrier gas.

The individual compounds were identiﬁed by MS and their identity was conﬁrmed by comparing their Kováts Index (KI), their mass spectra and retention times with those of authentic samples or with data already available in the NIST library and in the literature (Adams, 2007).

The KI of a compound is a number obtained by interpolation, relating the adjusted retention time of the sample compound to the adjusted retention times of two standards (in our analysis C8 and C32) eluted before and after the sample compound peak.

To evaluate the antibacterial and antifungal activity of essential oil of C. ladanifer, tested bacteria include four strains in which Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. These strains were collected from samples of different pathological origins, from patients consulting in the regional hospital of Meknes city. The tested fungi, included Candida albicans, Trichophyton rubrum and Aspergillus niger which were collected from the Military Hospital of Meknes city.

Inocula preparation

Before use, bacterial strains were revivified by subcultures in Luria-Bertani (LB) plates at 37°C for 24h. Stock bacterial inocula suspensions were obtained from 18 hours culture on Mueller Hinton broth at 37°C. Those final suspensions served for the inocula preparation. The cell density of each suspension was determined by using a counting chamber, and then adjusted to a concentration of 105-106 CFU/mL by dilution with Mueller Hinton broth.

Revivification of fungal was made by subcultures in malt extract agar plates (malt extract 30g/L and agar 20g/L) at 25°C for 7 days. After incubation, their spores were harvested by scraping the culture surface in sterile NaCl solution 0.9%. Then the spore suspension was concentrated by centrifugation at 10000g for 15 min at 4°C until a concentration of 106 spores/mL (counted with a hemocytometer). While, yeast strains were inoculated in yeast peptone-glucose agar (YPG) and incubated at 30°C for 48 h.

niger and T. rubrum were grown on Sabouraud agar at 35°C for 7 days, to induce conidia formations. Then, the culture was washed with 2 mL of saline solution (0.9% NaCl) and the suspension was transferred to a sterile tube, where the heavy particles were allowed to settle for 5 min. The upper homogeneous suspensions were transferred with a pipette Pasteur to a new sterile tube and filled with Sabouraud broth, then vortexed for few seconds. The mixture of conidial and hyphal fragments was ﬁltered with Wattman ﬁlter, which retains hyphal fragments and permits the passage of only T. rubrum and A. niger microconidia; this suspension is considered as the initial inoculum. After dilutions in Sabouraud broth, the inoculum was adjusted microscopically to about 104 CFU/mL.

Evaluation of antimicrobial activity by diffusion method in agar medium

The technique used is a modification of the method of Fauchère and Avril, (2002). It consists of depositing sterile disks of filter paper (Whatman N° 40) 6 mm in diameter soaked with a decreasing concentration range of essential oil 20, 10 and 5μL solubilized in the Tween 20% Erreur de traductiondeposited on a microbial mat and then measure the area where microorganisms could not develop.

A disk soaked with 20 μL Tween 20% was used as negative control on the same plate; Gentamicin® was used as a standard antibiotic for bacterial strains with a concentration of 0.04 g/mL Erreur de traductionand Canaflucon (Fluconazole)Win® capsule 150 mg with a concentration of 0.6 μg/mL was used as a positive control for fungal strains.

Erreur de traductionThe dishes are left for 1 hour at room temperature to allow the diffusion of the essential oil and are then incubated at 37°C, for 18 to 24 hours for the bacterial strains and 48 hours for the fungal strains. After incubation, the inhibition diameter, translating the antimicrobial activity of the essential oil, is thus determined as a translucent halo around the disc which is measured in millimeters, disc included.

Determination of minimum inhibitory concentration against bacteria

A microdilution of the tested essential oil is produced in a microplate containing 20 μL of Tween 20%, so as to generate a base dilution range 2. The concentration range is then produced in the 96 wells (Greiner, VWR).

20 μL of the essential oil is added to the first well of each line from which a basic geometric dilution 2 is carried out. Then 160 μL of Mueller Hinton broth (MHB) inoculated with 20 μL of a bacterial suspension at 106 CFU/mL; are added to each well. Each row is reserved for a particular strain, this work was carried out on four strains and therefore on four rows. Wells containing MHB inoculated with the determined strain are used as a positive control; those containing Tween 20% and non-inoculated MHB are used as a negative control. After an incubation time of 18h, the minimum inhibitory concentration (MIC) of the essential oil is deduced from the first well of the range devoid of microbial growth. The reading is carried out using a 2,3,5-diphenyltetrazolium chloride [TTC, red tetrazolium] colored indicator diluted in sterile distilled water to the order of 0.2 g/mL, by addition of 40 μL of TTC followed by an incubation for 30 min at 37°C, TTC reveals the presence of living bacteria by the appearance of a red coloration (Eloff, 1998).

To investigate the antifungal activity of the studied essential oil against A. niger and T. rubrum a modified microdilution technique described by Daouk et al., (1995) was used. Firstly, 50 μl of malt extract broth were added from the second to the 12th well. The essential oil was diluted in Tween 20 % (v/v) at a final concentration of 40 % (v/v), then 100 µL of this solution were deposed in the first well. Afterwards, scalar dilution was made by transferring 50 µL from the 1st to the 11th well. The 12th well was considered as growth control. Thereafter 50 μL of the fungal spore suspension was added to each well to reach a final concentration of 106 spores/mL. The microplate was sealed and incubated for 72 h at 30°C. The lowest essential oil concentration that prevents visible fungal growth was defined as the MIC. Likewise, the MIC determination against C. albicans, was performed in 96 well-microplates according to the protocol previously described with slight modifications of Chraibi et al. (2016). The essential oil was also serially diluted in YPG broth supplemented with agar at 0.15% (w/v). The 12th well was also considered as growth control. Then, 50 μL of fungal inoculum were added to each well at a final concentration of 103 CFU/mL. Finally, the microplate was sealed and incubated at 30°C for 48 h. Experiments were carried out in triplicate. Similarly, the lowest essential oil concentration that prevents visible fungal growth was defined as the MIC.

MIC was detected by lack of visual turbidity (matching the negative growth control). Subcultures were made from the clear wells which did not show any growth after incubation during the MIC assays on Mueller Hinton agar for bacteria and Sabouraud agar for fungi. Minimum Bactericidal Concentration (MBC) or Minimal Fungicidal Concentration (MFC) is calculated by transporting 10 μL of the wells showing no microbial growth on a solid medium. The lowest concentration of an antibacterial drug results in a 99.9% reduction of the initial microbial density. EP routinely performs antimicrobial effectiveness testing services which normally includes MBC (minimum bactericidal concentration)/MFC (minimum fungicidal concentration) in conjunction with MIC (minimum inhibitory concentration) to distinguish if antimicrobial compounds are bactericidal or bacteriostatic (Bassole et al., 2001).

RESULTS

Chemical analysis

The yield of essential oil obtained by distillation of fresh material ranged from 0,1-0,2% (mL per 200 g of fresh material). Twenty-eight volatile components of essential oil of C. ladanifer, were identiﬁed and quantiﬁed. Figure 1 shows the chromatogram of C. ladanifer oil on a DB-5 column, while Table 1 lists Kováts retention indices and percentage composition of the compounds which have been identiﬁed in this oil. A total of 28 compounds, amounting to 88.67% of the oil, were identiﬁed. Mono- and sesquiterpene hydrocarbons represented the most important fraction of the total oil composition (44.45%), while of the six monoterpene hydrocarbons identiﬁed (32.05%), the major ones were Verticiol (18.16%), camphene (17.70%), n-Butylcyclohexane (5.95%) and 3-Carene (5.23%). The 6 sesquiterpene hydrocarbons were relatively low (12.4%) with γ-Gurjunene (7.15%) being the major component. The oxygenated mono- and sesquiterpenoids made up an appreciable fraction of the oil (15.84%). Isolongifolol was the major component of the Oxygenated sesquiterpenes (1.41%), while of the 9 oxygenated monoterpenes identiﬁed (13.27%), the most important ones were cis-Sabinol (5.14%), trans-Sabinene hydrate (1.85%) and 1-p-Menthen-9-al (1.52%).

In this work, antimicrobial activity of essential oil of C. ladanifer against four bacterial strains and three fungal strains, known to cause several infections in humans, was evaluated in vitro. The bacteria tested E.c, P.a, S.e show a zone of inhibition lower than that detected by the antibiogram CFM (Ben Sassi et al., 2007; Mohammedi and Atik, 2011).These strains are resistant to gentamycin, but S.a is sensitive to it since it has a zone of inhibition of 23 mm which is greater than 20 mm.The results of the sensitivity test, of microbial strains to essential oil of C. ladanifer by the aromatogramme method (Table 2), show inhibition of bacterial growth proportional to its concentration, which is related to the diameter of the zone of inhibition.

Table 2 Inhibition zones caused by the essential oil in mm of C. ladanifer at different concentrations.

Analyzing the results of the aromatogram of the essential oil of this plant, we note that all the diameters of inhibition are greater or equal to 9 mm for all the concentrations used and for all the bacteria tested. These results are in favor of an antibacterial capacity of this plant, by referring to the reading established by Meena and Sethi, (1994) and Ponce et al. (2003). The diameter of the inhibition halos is noted as: not sensitive for diameters less than 8 mm; sensitive, for diameters 9-14 mm; very sensitive for diameters 15-19 mm, and extremely sensitive for diameters larger than 20 mm. Thus, Staphylococcus aureus shows extreme sensitivity, and the three other bacteria respond very positively to its antibacterial effect, since they are classed in Gram positive or Gram-negative bacteria, and they are already have innate or acquired resistance to antibiotics. It should be noted that these bacteria selected in this study are the most incriminated in the vast majority of human infectious diseases, nosocomial infections and food poisoning. The continuous evolution of their resistance is one of the main causes of anti-biotherapy failure. Their eradication by the essential oil of C. ladanifer is a therapeutic success and is in fact a possible opportunity to disappoint treatment to antibiotics.

These results are consistent with those found in literature, some studies have found significant antibacterial activity of C. ladanifer on strains of S. epidermidis and S. aureus than on Gram negative strains like E. coli and P. aeruginosa which are among the most difficult germs to treat clinically (Friedman et al., 2002; Burt, 2004). The intrinsic resistance to antibiotics makes them little vulnerable to the action of most antibacterial agents of natural or synthetic origin (Lomovskaya et al., 2007).

This study also shows that all doses essential oil of C. ladanifer showed an inhibitory capacity of mycelial growth as well as spore production. The diameter of zone of inhibition vary between 38 mm in C. albicans and 28 mm in T. rubrum, which is greater than the values of reference used here. Our results on fungal strains are in agreement with the work of Barros et al., (2013) and Greche et al. (2009), who showed a strong antifungal activity of C. ladanifer on some fungal strains.

Microdilution method

The minimum inhibitory concentration (MIC) values revealed that this oil was effectiveness against the tested bacteria and fungi and confirmed that it has broad spectrum antimicrobial effect.

The antibacterial effect unveiled seems to be strain dependent. In fact, strong inhibitory effect has been shown against all Gram-positive bacteria, especially S. epidermidis and S. aureus, since they were inhibited by very low concentrations 6.25 and 12.5 mg/mL (v/v) respectively. E. coli and P. aeruginosa are also sensible, but the MIC value is slightly greater 25 mg/mL (v/v). Previous studies confirmed that this latter bacterial strain was the most resistant (Ghannadi et al., 2012; Boukhatem et al., 2013; Haloui et al., 2015). Furthermore, concerning the antifungal activity against molds and yeasts (Table3), the screening test revealed that all tested fungal strains were sensitive to the C. ladanifer essential oil. Indeed, the strongest inhibitory effect was exerted against yeasts and molds (C. albicans and A. niger) with MIC values of 0.001 mg/mL (v/v). These results are in agreement with those of previous works (Hassane et al., 2011; Boukhatem et al., 2013). As regards to the fungal (T. rubrum) they were inhibited with MIC value of 0,01 mg/mL (v/v). The broad spectrum and the significant antimicrobial activity of the tested essential oil may be attributed to the synergistic interaction between the various components of this oil. In fact, it has been reported in previous studies that the inhibitory activity of an essential oil results from a complex interaction between its different constituents (Burt, 2004; Xianfei et al., 2007; Viuda-Martos et al., 2008).

Essential oils, extracted from plants of the genus Cistus sp, are known for their complex chemical composition and diversity of their constituents. They contain a large number of molecules, often present in trace and very close chemical polarity. These two characteristics complicate the separation and identification of these same molecules (Weyersthal et al., 1998).

Also, the share of unidentified compounds in the essential oils of Cistus sp, is generally not negligible. The chemical composition of the essential oil of C. ladanifer is chemotype Verticiol (18.16%), Camphene (17.70%), γ-Gurjunene (7.15%), n-Butylcyclohexane (5.95%) and Bornyl acetate (5.86%). These constituents are common to most essential oils of C. Ladanifer (Mariotti et al., 1997). Oxygen molecules, which are part of the composition of essential oils, are generally more active than hydrocarbon molecules, known for their poor antibacterial properties (Kalemba and Kunicka, 2003).

The hydrocarbon terpenes of the essential oil of C. ladanifer are also inactive. In contrast, oxygenated molecules inhibit the growth of most bacterial strains. The activity of the oxygenated molecules of essential oils depends both on the lipophilic nature of their hydrocarbon backbone and the hydrophilic nature of their functional groups (Kalemba and Kunicka, 2003). Molecules belonging to different chemical classes will therefore not produce the same effects according to the bacterial strains tested. Phenols are, because of the acidity of their hydroxyl substituent, described as the most active compounds.

Carbonyl compounds, functionalized with aldehyde or ketone groups, and alcohols are also recognized for their antibacterial properties (Kalemba and Kunicka, 2003). These two classes of molecules are part of the chemical composition of the essential oil of C. ladanifer. However, only the class of terpene alcohols has proved to be active. Gram negative bacteria are also very sensitive to the action of this class of molecules.

Bouamama et al. (2006) reported MIC values of crude extracts of C. villosus and C. monspeliensis against S. aureus of 0.76 and 25 mg/mL, respectively, and of 3.125 to 50 mg/mL for P. aeruginosa. Considering the antimicrobial activity of C. ladanifer, our work showed better results than C. monspeliensis against S. aureus and then the above both Cistus species against P. aeruginosa.

In this study, the minimal bactericidal concentration (CMB) and the minimum fungicidal concentration (CMF) were evaluated for the essential oil of C. ladanifer. The CMB/MIC and CMF / MIC ratios of C. ladanifer oil are equal to one for all microbial strains studied. This essential oil therefore seems to have a bactericidal and fungicidal action against all the bacterial and fungal strains tested. Table 3 shows the CMB and CMF of C. ladanifer essential oil against the tested germs.

Table 3 MIC, MBC and MFC values of essential oil of C. ladanifer.

Microbial strains

MIC (mg/mL)

MBC(mg/mL)

MFC(mg/mL)

MBC/MIC

MFC/MIC

E. coli

25

25

–

1

P. aeruginosa

25

25

–

1

S. epidermidis

12,5

12,5

–

1

S. aureus

6,25

6,25

–

1

A. niger

0,001

–

0,001

1

T. rubrum

0,01

–

0,01

1

C. albicans

0,001

–

0,001

1

CONCLUSION

The findings highlighted that C. ladanifer essential oil was able to inhibit the growth of a wide spectrum of microbial strains, known for their implications in human and animal infections. Its content on several antimicrobial active compounds and their likely interactions explained well its bioactivity. Hence, this essential oil can be a promising agent to control microbial growth, even if more detailed reports on its toxicity and mechanisms of action are requested to overcome the impediment of its application in several industries.